Genomic imprinting is a mammalian-specific phenomenon whereby the expression of a subset of genes is dependent on parental origin of the allele. Monoallelic expression is generally correlated with differential methylation of the parental alleles. Differences in methylation can be inherited as a primary imprinting mark from the gametes or acquired secondarily as a post-fertilization event. The significance of establishing the appropriate methylation pattern and achieving imprinted expression is illustrated by the fact that in the absence of parent of origin-specific expression, developmental defects ensue. Human diseases such as Prader-Willi, Angelman and Beckwith-Wiedemann syndromes result from the inappropriate expression of imprinted genes. The objective of this research is to enhance the understanding of how imprinted expression is achieved via an analysis of methylation acquisition at imprinted loci. The first goal is to conduct a temporal analysis of the acquisition of methylation during male gametogenesis at the paternally-methylated Gtl2 and Rasgrfl loci. Second, the role of DNA methyltransferase 3L in establishing methylation at imprinted loci during male gametogenesis will be examined. Finally, the post-fertilization establishment of methylation at the Gtl2 promoter and its relationship to monoallelic expression will be investigated. Bisulfite mutagenesis and DNA sequencing will be the primary methodology utilized to examine methylation in these analyses. This research will advance the field of imprinting by determining whether the establishment of imprinting marks is globally or independently controlled. Furthermore, it will test the hypothesis that DNA methyltransferase 3L is the enzyme responsible for methylation establishment in the male germline. In addition, this project will enhance undergraduate education by providing students the opportunity to conduct independent research in molecular genetics.